2.1 ENZYMATIC CONVERSION OF BLOOD TYPE
Based on the presence or absence of defined antigens, human blood may be classified into four main types, or groups, designated O, A, B, and AB. There are three major recognized subtypes of blood type A, known as A.sub.1, A.sub.int, and A.sub.2.
The carbohydrate structures associated with A.sub.1, A.sub.2, B and O blood types are shown in FIGS. 1A-1D. While A.sub.2 and B antigens consist of a single, external, antigenic component, the A.sub.1 antigen comprises two antigenic components, the major component having an external residue (FIG. 1A) and the minor component having both an external as well as an internal residue (FIG. 1A), relative to the carbohydrate chain.
Individuals with type A red cells have, in their plasma, antibodies directed against type B red cells (anti-B antibodies). Conversely, individuals with type B red cells have anti-A antibodies in their plasma. Persons with type O blood have antibodies directed toward both A and B antigens.
The presence of such antibodies makes blood transfusions problematic. If the host to a transfusion carries antibodies against the donor blood, a severe and potentially life-threatening reaction can result. The only blood type that can be safely transfused into persons of all blood types is type O blood, which is often referred to as "universal donor" blood. However, the availability of type O blood is insufficient to meet transfusion needs, because less than half of the population has type O blood.
Moreover, as a result of the limited shelf-life of donated blood, a disparity between the supply of blood available and transfusion needs often leads to the destruction of large quantities of blood stored in blood banks internationally.
In order to satisfy the demand for safely transfusable blood, and to more efficiently utilize the donated blood supply, technology has been developed which converts erythrocytes which are type A, B, or AB to "universal donor" blood.
Conversion of blood type B to type O may be accomplished using .alpha.-galactosidase enzyme originating from green coffee bean ("B-zyme"), which cleaves at the .alpha.1,3 bond linking the terminal galactose to a carbohydrate structure identical to the H-antigen associated with type O blood (cleavage indicated by a dotted line in FIG. 1C). Blood converted by this method has been safely transfused into patients (see, for example, U.S. Pat. Nos. 4,330,619 and 4,427,777; Lenny et al., 1991, Blood 77:1383-1388; Goldstein, 1989, Transfusion Medicine Reviews III(3):206-212). The coffee bean .alpha.-galactosidase gene has been cloned, characterized, and expressed to produce recombinant enzyme for use in the conversion of type B erythrocytes (Zhu and Goldstein, 1994, Gene 140:227-231).
Likewise, type A.sub.int -A.sub.2 blood has been successfully deantigenized using .alpha.-N-acetyl-galactosaminidase enzyme originating in chicken liver ("A-zyme"; U.S. Pat. No. 4,609,627; Goldstein et al., 1984, "Enzymatic Removal of Group A Antigens" in Abstracts of the 18th Congress of the ISBT, Karger, Munich, p. 86; Goldstein, 1989, Transfusion Medicine Reviews III(3):206-212). The chicken liver .alpha.-N-acetylgalactosaminidase gene has been cloned, characterized, and expressed (Zhu and Goldstein, 1993, Gene 137:309-314).
Because the A.sub.1 antigen comprises an internal as well as an external antigenic component, even after treatment with .alpha.-N-acetylgalactosaminidase, internal antigen remains. An endo-.beta.-galactosidase is required to remove the internal antigen. Prior to the present invention, however, an enzyme having the specificity, efficiency, and purity necessary for conversion of type A.sub.1 erythrocytes, on a scale commensurate with transfusion requirements in the United States and internationally, has been unavailable.